1. Field of the Invention
The invention relates to a new process for preparing sulfides labeled with sulfur-35, and for preparing amino acids and their derivatives labeled with sulfur-35, with high specific activity.
2. Description of the Background
The sulfur-containing amino acids L-cysteine and L-methionine play an important role in many biological processes, and in particular are incorporated in very varied proteins. The initiation of the biosynthesis of a polypeptide chain requires the presence of L-methionine; L-cysteine is necessary in the formation of disulfide bridges which stabilize the tertiary structure of proteins and also intervene at the active sites of many enzymes.
Sulfur-containing amino acids labeled with sulfur-35, and in particular methionine, are therefore very frequently used for the study of biological processes. In order to be used, it is necessary to have available products with a high specific activity, greater than 29.6 TBq to 37 TBq/mmole (800 to 1000 Ci/mmole)o
Two types of processes are known which make it possible to obtain L-cysteine and L-methionine labeled with sulfur-35: the chemical route and the microbiological route.
Methods of synthesis by the chemical route are currently little used, for their yield is low; they require a lot of equipment, many stages of handling, and are not suited to the handling of very small quantities of reagents which are employed for the manufacture of a final product of very high specific activity.
For example, the method described in the publication Heise and Mittag [Kernenergie, 8, 181-184 (1965)] produces (.sup.35 S) L-methionine with a specific activity of 2775 MBq/mmole (75 mCi/mmole).
L-Cysteine and L-methionine labeled with sulfur 35 which is currently commercially available are prepared by the microbiological route.
Several publications describe this preparation method; the works of Albahari and Skakun-Todorovic [Journal of labelled compounds and Radiopharmaceuticals, XIV, 5, 727-733 (1978)], of Graham and Stanley [Analytical Biochemistry 47, 505-513, (1972)], of Bretscher and Smith [Analytical Biochemistry, 47, 310-312, (1972)] may be mentioned as examples.
Microorganisms (yeasts or bacteria) are cultured in minimum medium, the only source of sulfur being supplied by sodium or ammonium sulfate, labeled with sulfur-35, with a high specific activity.
The sulfur-35 is incorporated in the proteins, which are then hydrolyzed and the sulfur-containing amino acids are purified, from the hydrolysate by chromatography. This chromatography stage does not, however, manage to totally remove the contamination by other, unlabeled amino acids, (for example L-valine or L-leucine), as well as by (.sup.35 S)-D-methionine
Each of these contaminants represents 2 to 3% of the (.sup.35 S)-L-methionine preparation obtained.
On the other hand, this method of preparation is relatively lengthy; it takes at least 24 hours for the sulfur to be incorporated in the proteins; another delay of 24 hours is necessary for the hydrolysis of the proteins to be complete.
The preparations of amino acids labeled with sulfur-35 must be frequently replaced, for the half-life of sulfur-35 is relatively short, and (.sup.35 S)-L-methionine has little chemical stability and is very rapidly oxidized to L-methionine sulfoxide.
It is thus particularly desirable to have available a simple and easy method of use which makes it possible to prepare these compounds as and when required.
The Inventors, in seeking to solve this problem, have directed their work towards the development of a method of enzymatic synthesis.
Various metabolical routes which lead to the synthesis of sulfur-containing amino acids are known, as well as the enzymes which intervene in each of the stages of this synthesis: for a general review, see, for example, Methods in Enzymology, volume 143, (1987), Editors W. B. Jakoby and Griffith.
The organic sulfur of cysteine and methionine arises from inorganic sulfur which can be taken up by certain microorganisms and reduced, in fine to the sulfide form. The sulfides react with serine or homoserine (or their O-acyl derivatives) to give cysteine and homocysteine.
Various enzymes can catalyze this reaction: [for a detailed review, cf. Soda, Methods in Enzymology 143 453-457 (1987)].
For example, O-acetylserine sulfhydrylase (EC.4.2.99.8), cystathionine .beta.-synthase (EC.4.2.1.22), O-acetylhomoserine (thiol)-lyase (EC.4.2.99.10), and so on, may be mentioned.
Although these enzymes are known and have been purified, their employment in the preparation of amino acids labeled with sulfur-35, and with a high specific activity, has never been proposed.
Indeed, although the reactions catalyzed by these enzymes can be easily carried out in vitro in the presence of substrates which are not radio-labeled, and when there are no particular requirements regarding reaction yield or the quality of the final product, the same does not apply, however, when these reactions must be carried out in the presence of radio-labeled substrates and while ensuring a high specific activity in the final product as well as a high chemical and radiochemical purity.
The problems to be solved in order to achieve this aim are many because it is necessary to limit as much as possible the number of necessary stages, each stage having, in addition, to be compatible with the preceding and following stages.
The principal problem which has to be solved in order to allow the use of the enzymatic reaction with the best results is that of the employment of a suitable labeled substrate with high specific activity.
The problem does not arise in the methods for preparing amino acids labeled by the bacteriological route, because the sulfur-35 used in these methods, obtained from the irradiation of potassium chloride according to the reaction .sup.35 Cl(n,p).sup.35 S, is purified in the form of sulfate which can be taken up by bacteria.
However, the enzymes which synthesize the sulfur containing amino acids use sulfides as substrate and not sulfates. It is thus necessary to carry out, prior to the enzymatic reaction, the reduction of the sulfate to sulfide. However, this stage can lead to a significant reduction in the specific activity.
The inventors have now selected a reducing mixture and developed reaction conditions which allow the reduction of the sulfate to sulfide with a good yield, and without loss of specific activity.